The Cl- transporter ClC-7 is essential for phagocytic clearance by microglia.

Microglia, professional phagocytic cells of the brain, rely upon the appropriate activation of lysosomes to execute their immune and clearance functions. Lysosomal activity is, in turn, modulated by a complex network of over 200 membrane and accessory proteins that relay extracellular cues to these key degradation centers. The ClC-7 chloride (Cl-)-proton (H+) antiporter (also known as CLCN7) is localized to the endolysosomal compartments and mutations in CLCN7 lead to osteopetrosis and neurodegeneration. Although the functions of ClC-7 have been extensively investigated in osteoclasts and neurons, its role in microglia in vivo remains largely unexamined. Here, we show that microglia and embryonic macrophages in zebrafish clcn7 mutants cannot effectively process extracellular debris in the form of apoptotic cells and ß-amyloid. Despite these functional defects, microglia develop normally in clcn7 mutants and display normal expression of endosomal and lysosomal markers. We also find that mutants for ostm1, which encodes the ß-subunit of ClC-7, have a phenotype that is strikingly similar to that of clcn7 mutants. Together, our observations uncover a previously unappreciated role of ClC-7 in microglia and contribute to the understanding of the neurodegenerative phenotypes that accompany mutations in this channel.

Oligodendrocyte development and myelin sheath formation are regulated by the antagonistic interaction between the Rag-Ragulator complex and TFEB.

Myelination by oligodendrocytes is critical for fast axonal conduction and for the support and survival of neurons in the central nervous system. Recent studies have emphasized that myelination is plastic and that new myelin is formed throughout life. Nonetheless, the mechanisms that regulate the number, length, and location of myelin sheaths formed by individual oligodendrocytes are incompletely understood. Previous work showed that the lysosomal transcription factor TFEB represses myelination by oligodendrocytes and that the RagA GTPase inhibits TFEB, but the step or steps of myelination in which TFEB plays a role have remained unclear. Here, we show that TFEB regulates oligodendrocyte differentiation and also controls the length of myelin sheaths formed by individual oligodendrocytes. In the dorsal spinal cord of tfeb mutants, individual oligodendrocytes produce myelin sheaths that are longer than those produced by wildtype cells. Transmission electron microscopy shows that there are more myelinated axons in the dorsal spinal cord of tfeb mutants than in wildtype animals, but no significant change in axon diameter. In contrast to tfeb mutants, oligodendrocytes in rraga mutants produce shorter myelin sheaths. The sheath length in rraga; tfeb double mutants is not significantly different from wildtype, consistent with the antagonistic interaction between RagA and TFEB. Finally, we find that the GTPase activating protein Flcn and the RagCa and RagCb GTPases are also necessary for myelination by oligodendrocytes. These findings demonstrate that TFEB coordinates myelin sheath length and number during myelin formation in the central nervous system.

Unmyelinated sensory neurons use Neuregulin signals to promote myelination of interneurons in the CNS.

The signaling mechanisms neurons use to modulate myelination of circuits in the central nervous system (CNS) are only partly understood. Through analysis of isoform-specific neuregulin1 (nrg1) mutants in zebrafish, we demonstrate that nrg1 type II is an important regulator of myelination of two classes of spinal cord interneurons. Surprisingly, nrg1 type II expression is prominent in unmyelinated Rohon-Beard sensory neurons, whereas myelination of neighboring interneurons is reduced in nrg1 type II mutants. Cell-type-specific loss-of-function studies indicate that nrg1 type II is required in Rohon-Beard neurons to signal to other neurons, not oligodendrocytes, to modulate spinal cord myelination. Together, our data support a model in which unmyelinated neurons express Nrg1 type II proteins to regulate myelination of neighboring neurons, a mode of action that may coordinate the functions of unmyelinated and myelinated neurons in the CNS.

Promoting validation and cross-phylogenetic integration in model organism research.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.

A lysosomal regulatory circuit essential for the development and function of microglia.

As the primary phagocytic cells of the central nervous system, microglia exquisitely regulate their lysosomal activity to facilitate brain development and homeostasis. However, mechanisms that coordinate lysosomal activity with microglia development, chemotaxis, and function remain unclear. Here, we show that embryonic macrophages require the lysosomal guanosine triphosphatase (GTPase) RagA and the GTPase-activating protein Folliculin to colonize the brain in zebrafish. We demonstrate that embryonic macrophages in rraga mutants show increased expression of lysosomal genes but display significant down-regulation of immune- and chemotaxis-related genes. Furthermore, we find that RagA and Folliculin repress the key lysosomal transcription factor Tfeb and its homologs Tfe3a and Tfe3b in the macrophage lineage. Using RNA sequencing, we establish that Tfeb and Tfe3 are required for activation of lysosomal target genes under conditions of stress but not for basal expression of lysosomal pathways. Collectively, our data define a lysosomal regulatory circuit essential for macrophage development and function in vivo.

A cAMP Sensor Based on Ligand-Dependent Protein Stabilization.

cAMP is a ubiquitous second messenger with many functions in diverse organisms. Current cAMP sensors, including Föster resonance energy transfer (FRET)-based and single-wavelength-based sensors, allow for real time visualization of this small molecule in cultured cells and in some cases in vivo. Nonetheless the observation of cAMP in living animals is still difficult, typically requiring specialized microscopes and ex vivo tissue processing. Here we used ligand-dependent protein stabilization to create a new cAMP sensor. This sensor allows specific and sensitive detection of cAMP in living zebrafish embryos, which may enable new understanding of the functions of cAMP in living vertebrates.

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Basic science under threat: Lessons from the Skirball Institute.

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises.

Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models.

Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

Myelination induces axonal hotspots of synaptic vesicle fusion that promote sheath growth.

Myelination of axons by oligodendrocytes enables fast saltatory conduction. Oligodendrocytes are responsive to neuronal activity, which has been shown to induce changes to myelin sheaths, potentially to optimize conduction and neural circuit function. However, the cellular bases of activity-regulated myelination in vivo are unclear, partly due to the difficulty of analyzing individual myelinated axons over time. Activity-regulated myelination occurs in specific neuronal subtypes and can be mediated by synaptic vesicle fusion, but several questions remain: it is unclear whether vesicular fusion occurs stochastically along axons or in discrete hotspots during myelination and whether vesicular fusion regulates myelin targeting, formation, and/or growth. It is also unclear why some neurons, but not others, exhibit activity-regulated myelination. Here, we imaged synaptic vesicle fusion in individual neurons in living zebrafish and documented robust vesicular fusion along axons during myelination. Surprisingly, we found that axonal vesicular fusion increased upon and required myelination. We found that axonal vesicular fusion was enriched in hotspots, namely the heminodal non-myelinated domains into which sheaths grew. Blocking vesicular fusion reduced the stable formation and growth of myelin sheaths, and chemogenetically stimulating neuronal activity promoted sheath growth. Finally, we observed high levels of axonal vesicular fusion only in neuronal subtypes that exhibit activity-regulated myelination. Our results identify a novel "feedforward" mechanism whereby the process of myelination promotes the neuronal activity-regulated signal, vesicular fusion that, in turn, consolidates sheath growth along specific axons selected for myelination.

The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity.

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.

The Lysosomal Transcription Factor TFEB Represses Myelination Downstream of the Rag-Ragulator Complex.

Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga-/- mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb-/- single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.

Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process. VIDEO ABSTRACT.

Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta.

Mural cells (vascular smooth muscle cells and pericytes) play an essential role in the development of the vasculature, promoting vascular quiescence and long-term vessel stabilization through their interactions with endothelial cells. However, the mechanistic details of how mural cells stabilize vessels are not fully understood. We have examined the emergence and functional role of mural cells investing the dorsal aorta during early development using the zebrafish. Consistent with previous literature, our data suggest that cells ensheathing the dorsal aorta emerge from a sub-population of cells in the adjacent sclerotome. Inhibition of mural cell recruitment to the dorsal aorta through disruption of pdgfr signaling leads to a reduced vascular basement membrane, which in turn results in enhanced dorsal aorta vessel elasticity and failure to restrict aortic diameter. Our results provide direct in vivo evidence for a functional role for mural cells in patterning and stabilization of the early vasculature through production and maintenance of the vascular basement membrane to prevent abnormal aortic expansion and elasticity.

Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

Individual Neuronal Subtypes Exhibit Diversity in CNS Myelination Mediated by Synaptic Vesicle Release.

Regulation of myelination by oligodendrocytes in the CNS has important consequences for higher-order nervous system function (e.g., [1-4]), and there is growing consensus that neuronal activity regulates CNS myelination (e.g., [5-9]) through local axon-oligodendrocyte synaptic-vesicle-release-mediated signaling [10-12]. Recent analyses have indicated that myelination along axons of distinct neuronal subtypes can differ [13, 14], but it is not known whether regulation of myelination by activity is common to all neuronal subtypes or only some. This limits insight into how specific neurons regulate their own conduction. Here, we use a novel fluorescent fusion protein reporter to study myelination along the axons of distinct neuronal subtypes over time in zebrafish. We find that the axons of reticulospinal and commissural primary ascending (CoPA) neurons are among the first myelinated in the zebrafish CNS. To investigate how activity regulates myelination by different neuronal subtypes, we express tetanus toxin (TeNT) in individual reticulospinal or CoPA neurons to prevent synaptic vesicle release. We find that the axons of individual tetanus toxin expressing reticulospinal neurons have fewer myelin sheaths than controls and that their myelin sheaths are 50% shorter than controls. In stark contrast, myelination along tetanus-toxin-expressing CoPA neuron axons is entirely normal. These results indicate that while some neuronal subtypes modulate myelination by synaptic vesicle release to a striking degree in vivo, others do not. These data have implications for our understanding of how different neurons regulate myelination and thus their own function within specific neuronal circuits.

The Rag-Ragulator Complex Regulates Lysosome Function and Phagocytic Flux in Microglia.

Microglia are resident macrophages of the CNS that are essential for phagocytosis of apoptotic neurons and weak synapses during development. We show that RagA and Lamtor4, two components of the Rag-Ragulator complex, are essential regulators of lysosomes in microglia. In zebrafish lacking RagA function, microglia exhibit an expanded lysosomal compartment, but they are unable to properly digest apoptotic neuronal debris. Previous biochemical studies have placed the Rag-Ragulator complex upstream of mTORC1 activation in response to cellular nutrient availability. Nonetheless, RagA and mTOR mutant zebrafish have distinct phenotypes, indicating that the Rag-Ragulator complex has functions independent of mTOR signaling. Our analysis reveals an essential role of the Rag-Ragulator complex in proper lysosome function and phagocytic flux in microglia.

A zinc finger protein that regulates oligodendrocyte specification, migration and myelination in zebrafish.

Precise control of oligodendrocyte migration and development is crucial for myelination of axons in the central nervous system (CNS), but important questions remain unanswered about the mechanisms controlling these processes. In a zebrafish screen for myelination mutants, we identified a mutation in zinc finger protein 16-like (znf16l). znf16l mutant larvae have reduced myelin basic protein (mbp) expression and reduced CNS myelin. Marker, time-lapse and ultrastructural studies indicated that oligodendrocyte specification, migration and myelination are disrupted in znf16l mutants. Transgenic studies indicated that znf16l acts autonomously in oligodendrocytes. Expression of Zfp488 from mouse rescued mbp expression in znf16l mutants, indicating that these homologs have overlapping functions. Our results defined the function of a new zinc finger protein with specific function in oligodendrocyte specification, migration and myelination in the developing CNS.